We have recently discovered that decreased expression of proteinase-3 (PR3), a serine protease, is associated with drug resistance. Inhibition of cellular PR3 expression by an antisense PR3 oligodeoxynucleotide resulted in a significant reduction fin reactive oxygen species (ROS) generation and drug-induced apoptosis, and increased cellular resistance to doxorubicin (DOX) or daunorubicin (DNR). In this application, we propose to explore the molecular mechanism(s) by which PR3 mediates drug-induced apoptosis and increases the efficacy of anticancer agents, and how its loss causes drug resistance. Our long-term objective is to apply the knowledge obtained from these studies to identify and/or develop novel and more effective cancer therapeutic regimens. The Specific Aims are to (1) determine whether increased PR3 expression by gene transfer experiments enhances the cytotoxic effects of DOX and DNR, and (2) investigate the role of the PR3 in mediating increased reactive oxygen species (ROS) and delineate the molecular mechanism(s) of PR3-mediated drug-induced apoptosis, and (3) determine whether the PR3-mediated drug-induced apoptosis is dependent on the proteolytic activity of PR3 by identifying the domain of PR3 with apoptotic function. Specific Aim 1 will (a) evaluate whether the increase in PR3 enhances DOX- or DNR-induced apoptosis in drug sensitive and resistant cells, (b) determine the role of PR3 in drug-induced ROS production and apoptosis, (c) examine the subcellular localization of PR3, and (d) ascertain biochemically how PR3 expression modulates the effect of DOX on triggering apoptosis. Specific Aim 2 will (a) determine whether the PR3-mediated increase in ROS occurs through a mitochondrion-dependent mechanism, and by increased activation of cellular tumor necrosis factor-a (TNFct), (b) investigate whether inhibition of PR3 expression blocks neutral sphingomyelinase (N-SMase) activation and ceramide generation, and whether increased expression of PR3 activates these processes, and (c) assess whether the PR3-mediated drug induced increase in ROS production induces ceramide generation, and activates c-Jun-Terminal Kinase (JNK) and AP1 transcription factor. In Specific Aim 3, we will (a) construct FLAG-tagged expression vectors containing regions of PR3 that, upon expression in cells, produce PR3 fragments which either contain or lack the full components of catalytic domain of PR3, (b) transfect cells lacking PR3 with these expression vectors, and identify a fragment of PR3 which enhances drug-induced apoptosis, (c) assess the effect of this PR3 fragment in modulating drug-induced apoptosis in the drug sensitive and resistant cell lines, (d) generate deletion mutants as well as perform site-directed mutagenesis studies to refine the domain of PR3 with apoptotic function, and (e) design peptides from this domain and examine whether they are able to modulate the cytotoxicity of DOX and DNR. These studies will aid in understanding how PR3 is capable of mediating drug-induced apoptosis, and will be useful for the development of more effective chemotherapeutic or potential gene therapy strategies.